Treponema pallidum triplet antigen

ABSTRACT

A  Treponema pallidum  triplet antigen construct is disclosed which includes three  Treponema pallidum  antigens (TP15, TP17, and TP47), as well as a ten amino acid leader sequence (tag 261) and human copper zinc superoxide dismutase (hSOD). This construct is optimized for in vitro diagnosis of syphilis infection. Plasmids containing DNA encoding the triplet antigen, host cells, production methods, detection methods, and kits are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/980,200, filed Dec. 28, 2015, which is a divisional of Ser. No.14/193,530, filed Feb. 28, 2014, which is a divisional of U.S.application Ser. No. 13/350,235, filed Jan. 13, 2012, now U.S. Pat. No.8,691,950, issued Apr. 8, 2014, which claims the benefit of U.S.Provisional Application No. 61/432,570, filed Jan. 13, 2011.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The invention relates to the field of recombinant antigens, and moreparticularly to a Treponema pallidum triplet antigen construct and itsuse in immunoassays for the detection of syphilis.

BACKGROUND OF THE INVENTION

Over the past half century, effective antibiotic treatment programs havemade syphilis relatively uncommon in the United States, with less than7,100 primary and secondary cases diagnosed in 2003. However, recentdata indicates that reported cases are again increasing in subsets ofthe population, and periodic epidemics of syphilis have occurred fordecades. In 1995, the number of new cases of syphilis worldwide wasestimated to be 12 million per year. If untreated, syphilis can evolvefrom localized primary lesions (chancres) to disseminated, chronicinfections, including secondary, latent, and tertiary forms.

As a syphilitic infection can produce a variable range of symptoms inhumans, laboratory tests are often required to definitively diagnose aninfection. Due to the inability to culture the causative organism,Treponema pallidum (T. pallidum)(TP), in vitro, a need exists for thedevelopment and optimization of in vitro methods for the detection of T.pallidum in diverse clinical specimens [Morse, Salud Publica Mex 5(Suppl45):S698-5708, 2003]. While enzyme-linked immunosorbent assays (ELISAs)for Treponema are commercially available, they exhibit varyingefficiencies at different disease stages [Schmidt et al., J ClinMicrobiol 38:1279-1282 (2000)]. Several ELISAs based on whole celllysate were developed which presented sensitivity of 93.3% to 100% andspecificity of 95.5% to 99.8% [Castro et al., J Clin Microbiol41:250-253 (2003)].

In recent years, several immunodominant and putatively pathogen-specificmembrane lipoproteins of T. pallidum have been identified in patientswith syphilis and in infants with congenital syphilis. These patientsand infants developed antigen specific antibodies which could bedetected by immunoblot and by enzyme immunoassay. Therefore, recentmethods of detection use recombinant antigens, mainly themembrane-integrated proteins 47 kDa, 17 kDa and 15 kDa (TP47, TP17, andTP15, respectively), in treponemal ELISA tests. Although TP47 was theearliest identified, as well as the most abundant and highly immunogenic[Norgard et al., Infect Immun 54:500-506 (1986)], the later identifiedTP15 and TP17, present in lower amounts, are also strongly immunogenic[Purcell et al., Infect Immun 57:3708-3714 (1989); Akins et al., InfectImmun 61:1202-1210 (1993)].

Given the increase in reported cases of syphilis and the periodicepidemics, as well as the severity of the disease, a need continues toexist for sensitive and specific immunoassays for detection of Treponemapallidum.

BRIEF SUMMARY OF THE INVENTION

To this end, the invention provides a recombinant plasmid encoding aTreponema pallidum triplet antigen. The plasmid comprises nucleic acidencoding an amino acid sequence selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, andSEQ ID NO:12.

The invention further provides a recombinant plasmid encoding aTreponema pallidum triplet antigen, the recombinant plasmid selectedfrom the group consisting of: the plasmid designated p261nS-TP17-15-47and deposited with the American Type Culture Collection (“ATCC”) as ATCCAccession No. PTA-11590 on Jan. 12, 2011; the plasmid designatedp261nS-TP47-17-15 and deposited with the American Type CultureCollection as ATCC Accession No. PTA-11589 on Jan. 12, 2011; the plasmiddesignated p261nS-TP17-47-15; the plasmid designated p261nS-TP47-15-17;the plasmid designated p261nS-TP15-17-47; and the plasmid designatedp261nS-TP15-47-17.

Vectors, host cells, and triplet antigen production methods using thehost cells are also provided.

Additionally, the invention provides the Treponema pallidum tripletantigen having an amino acid sequence selected from the group consistingof SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,and SEQ ID NO:12. A method of detecting the presence of Treponemapallidum antibodies in a sample is further provided, which uses theTreponema pallidum triplet antigen, as well as kits for such detection.

Additional features and advantages of the subject invention will beapparent from the description which follows when considered inconjunction with the attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the reaction scheme for the VITROS® Syphilis TPAtest;

FIG. 2 shows the plasmid map of pUC57-TP15;

FIG. 3 shows the plasmid map of pUC57-TP17;

FIG. 4 shows the plasmid map of pUC57-TP47;

FIG. 5 shows the plasmid map of pUC57-hSOD;

FIG. 6 shows the TP construct for cloning into pB10G;

FIG. 7 shows the hSOD construct for cloning into a pB10G tripletplasmid;

FIG. 8 shows the plasmid map of pB10G;

FIG. 9 shows the plasmid map of pB10G-TP(1);

FIG. 10 shows the plasmid map of pB10G-TP(2);

FIG. 11 shows the plasmid map of pB10G-TP(3);

FIG. 12 shows the TP(1) construct for cloning into pB10G-TP(2);

FIG. 13 shows the plasmid map of pB10G-TP(1-2);

FIG. 14 shows the TP(3) construct for cloning into pB10G-TP(1-2);

FIG. 15 shows the plasmid map of pB10G-TP(1-2-3);

FIG. 16 shows the plasmid map of pB10G-TP(1-2-3) with the uniquerestriction enzyme site;

FIG. 17 shows the plasmid map of p261nS-TP(1-2-3);

FIG. 18 illustrates the direct assay format according to the subjectinvention; and

FIG. 19 illustrates the indirect assay format according to the subjectinvention.

DETAILED DESCRIPTION OF THE INVENTION

The assay (detection method) of the subject invention uses recombinantTreponema pallidum (TP)(the causative agent of Syphilis) outer membraneprotein antigens to detect patient sample anti-IgG, anti-IgM, andanti-IgA antibodies. The recombinant protein antigens of interest are a15 kilodalton antigen (TP15), a 17 kilodalton antigen (TP17), and a 47kilodalton antigen (TP47). A fused recombinant antigen construct hasbeen developed which incorporates the three antigens of interest as wellas human copper zinc superoxide dismutase (hSOD). In addition to theTreponema pallidum antigenic sequences and the hSOD, a 10 amino acid tag(the “261sequence”) is present at the N-terminus of the fused antigenconstruct to facilitate evaluation by Western blot and ELISA, and toprovide a means for affinity purification if desired. The assay of thesubject invention uses this fused recombinant antigen construct.

In one embodiment, the assay is the VITROS® Syphilis TPA test and theassay is performed using the VITROS® ECi/ECiQ Immunodiagnostic Systems,VITROS® 3600 Immunodiagnostic System, or VITROS® 5600 Integrated Systemusing Intellicheck® Technology. Each of these analyzers is availablefrom Ortho-Clinical Diagnostics, Inc. (OCD), 100 Indigo Creek Drive,Rochester, N.Y. 14626. Throughout this application, the use of thetrademark VITROS® refers to the line of chemistry and immunodiagnosticanalyzers and products commercially available from OCD. The use of thetrademark INTELLICHECK® refers to the technology commercially availablefrom OCD which monitors, verifies, and documents diagnostic checksthroughout sample and assay processing for accurate and efficient resultreporting. An immunometric immunoassay technique is used, which involvesthe reaction of IgG, IgM or IgA antibodies present in the sample with abiotinylated recombinant TP antigen and a horseradish peroxidase(HRP)-labeled recombinant TP antigen conjugate. The antibody-antigencomplex is captured by streptavidin on the wells (SAC wells). Unboundmaterials are removed by washing. The bound HRP conjugate is measured bya luminescent reaction. A reagent containing luminogenic substrate (aluminol derivative and a peracid salt) and an electron transfer agent isadded to the wells. The HRP in the bound conjugate catalyzes theoxidation of the luminol derivative, producing light. The electrontransfer agent (a substituted acetanilide) increases the level of lightproduced and prolongs its emission. The light signals are read by theanalyzer system. The bound HRP conjugate is directly proportional to theconcentration of anti-TP antibody present. This reaction scheme isillustrated in FIG. 1, where 10 represents the streptavidin coastedwell, 12 represents the biotinylated TP antigen, 14 represents theantibodies to TP antigens present in a sample, 16 represents HRP labeledTP antigen, 18 represents signal reagent with enhancer, and 20represents luminescence. Superoxide dismutase is present in the assaybiotin reagent formulation to block binding of anti-SOD antibodies thatmay be present in the patient sample. This prevents a false reactivesignal from being generated.

The invention provides a recombinant plasmid encoding a Treponemapallidum triplet antigen. The plasmid comprises nucleic acid encoding anamino acid sequence selected from the group consisting of SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

In one embodiment, the plasmid is designated p261nS-TP17-15-47 and isdeposited with the American Type Culture Collection (“ATCC”) as ATCCAccession No. PTA-11590. Plasmid p261nS-TP17-15-47 includes nucleic acidhaving the nucleotide sequence as shown in SEQ ID NO:3, encoding thetriplet antigen construct having the amino acid sequence as shown in SEQID NO:9. In this particular embodiment, amino acids 5-14 encode the 261tag, amino acids 17-150 encode TP17, amino acids 155-277 encode TP15,amino acids 282-695 encode TP47, and amino acids 697-849 encode hSOD.

In another embodiment, the plasmid is designated p261nS-TP47-17-15 andis deposited with the American Type Culture Collection as ATCC AccessionNo. PTA-11589. Plasmid p261nS-TP47-17-15 includes nucleic acid havingthe nucleotide sequence as shown in SEQ ID NO:6, encoding the tripletantigen construct having the amino acid sequence as shown in SEQ IDNO:12. In this particular embodiment, amino acids 5-14 encode the 261tag, amino acids 17-430 encode TP47, amino acids 435-568 encode TP17,amino acids 573-695 encode TP15, and amino acids 697-849 encode hSOD.

In a further embodiment, the plasmid is designated p261nS-TP17-47-15.Plasmid p261nS-TP17-47-15 includes nucleic acid having the nucleotidesequence as shown in SEQ ID NO:4, encoding the triplet antigen constructhaving the amino acid sequence as shown in SEQ ID NO:10. In thisparticular embodiment, amino acids 5-14 encode the 261 tag, amino acids17-150 encode TP17, amino acids 155-568 encode TP47, amino acids 571-693encode TP15, and amino acids 695-847 encode hSOD.

In yet another embodiment, the plasmid is designated p261nS-TP47-15-17.Plasmid p261nS-TP47-15-17 includes nucleic acid having the nucleotidesequence as shown in SEQ ID NO:5, encoding the triplet antigen constructhaving the amino acid sequence as shown in SEQ ID NO:11. In thisparticular embodiment, amino acids 5-14 encode the 261 tag, amino acids17-430 encode TP47, amino acids 435-557 encode TP15, amino acids 560-693encode TP17, and amino acids 695-847 encode hSOD.

In an additional embodiment, the plasmid is designatedp261nS-TP15-17-47. Plasmid p261nS-TP15-17-47 includes nucleic acidhaving the nucleotide sequence as shown in SEQ ID NO:1, encoding thetriplet antigen construct having the amino acid sequence as shown in SEQID NO:7. In this particular embodiment, amino acids 5-14 encode the 261tag, amino acids 17-139 encode TP15, amino acids 143-276 encode TP17,amino acids 281-694 encode TP47, and amino acids 696-848 encode hSOD.

In another additional embodiment, the plasmid is designatedp261nS-TP15-47-17. Plasmid p261nS-TP15-47-17 includes nucleic acidhaving the nucleotide sequence as shown in SEQ ID NO:2, encoding thetriplet antigen construct having the amino acid sequence as shown in SEQID NO:8. In this particular embodiment, amino acids 5-14 encode the 261tag, amino acids 17-139 encode TP15, amino acids 143-556 encode TP47,amino acids 559-692 encode TP17, and amino acids 694-846 encode hSOD.

The ATCC is located at 10801 University Boulevard, Manassas, Va.20110-2209 USA, and each of the above deposits was made on Jan. 12, 2011under the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure (the“Budapest Treaty”).

Each Treponema pallidum triplet antigen construct includes threeTreponema pallidum antigens (TP15, TP17, and TP47). While each has beendefined by its amino acid sequence as well as a nucleotide sequence, itshould be readily apparent that nucleotide additions, deletions, and/orsubstitutions, such as those which do not affect the translation of theDNA molecule, are within the scope of a particular nucleotide sequence(i.e. the amino acid sequence encoded thereby remains the same). Suchadditions, deletions, and/or substitutions can be, for example, theresult of point mutations made according to methods known to thoseskilled in the art. It is also possible to substitute a nucleotide whichalters the amino acid sequence encoded thereby, where the amino acidsubstituted is a conservative substitution or where amino acid homologyis conserved. It is also possible to have minor nucleotide additions,deletions, and/or substitutions which do not alter the function of theresulting triplet (i.e. its ability to detect anti-TP15, anti-TP17,and/or anti-TP47 antibodies).

Amino acid additions, deletions, and/or substitutions which do notnegate the ability of the resulting triplet to detect anti-TP15,anti-TP17, and/or anti-TP47 antibodies are thus within the scope of aparticular amino acid sequence. Such additions, deletions, and/orsubstitutions can be, for example, the result of point mutations in theDNA encoding the amino acid sequence, such point mutations madeaccording to methods known to those skilled in the art. Substitutionsmay be conservative substitutions of amino acids. Two amino acidresidues are conservative substitutions of one another, for example,where the two residues are of the same type. In this regard, proline,alanine, glycine, serine, and threonine, all of which are neutral,weakly hydrophobic residues, are of the same type. Glutamine, glutamicacid, asparagine, and aspartic acid, all of which are acidic,hydrophilic residues, are of the same type. Another type of residue isthe basic, hydrophilic amino acid residue, which includes histidine,lysine, and arginine. Leucine, isoleucine, valine, and methionine, allof which are hydrophobic, aliphatic amino acid residues, form yetanother type of residue. Yet another type of residue consists ofphenylalanine, tyrosine, and tryptophan, all of which are hydrophobic,aromatic residues. Further descriptions of the concept of conservativesubstitutions are given by French and Robson [J Molecular Evolution19:171-175 (1983)], Taylor [J Theor Biol 119:205-218 (1986)], and Bordoand Argos [J Mol Biol 217:721-729 (1991)].

While the presently preferred vector system for provision of the nucleicacid encoding the triplet antigen construct is a plasmid vector, othervector systems can also be used. Furthermore, while the presentlypreferred host cell for expression of the triplet antigen construct isthe bacterial host cell Escherichia coli, any suitable host and/orvector system can be used to express the triplet antigen construct.Other suitable bacterial hosts, yeasts hosts (such as Saccharomycescerevisiae), as well as mammalian (for example, Hela cells, Cv-1 cells,COS cells) and insect hosts (such as Drosophila cell lines), can beused.

Techniques for introducing the nucleic acid molecules into the hostcells may involve the use of expression vectors which comprise thenucleic acid molecules. These expression vectors (such as plasmids andviruses; viruses including bacteriophage) can then be used to introducethe nucleic acid molecules into the suitable host cells. For example,DNAencoding the triplet antigen can be injected into the nucleus of ahost cell or transformed into the host cell using a suitable vector, ormRNA encoding the triplet antigen can be injected directly into the hostcell, in order to obtain expression of triplet antigen in the host cell.

Various methods are known in the art for introducing nucleic acidmolecules into host cells. One method is microinjection, in which DNA isinjected directly into the nucleus of cells through fine glass needles(or RNA is injected directly into the cytoplasm of cells).Alternatively, DNA can be incubated with an inert carbohydrate polymer(dextran) to which a positively charged chemical group (DEAE, fordiethylaminoethyl) has been coupled. The DNA sticks to the DEAE-dextranvia its negatively charged phosphate groups. These large DNA-containingparticles stick in turn to the surfaces of cells, which are thought totake them in by a process known as endocytosis. Some of the DNA evadesdestruction in the cytoplasm of the cell and escapes to the nucleus,where it can be transcribed into RNA like any other gene in the cell. Inanother method, cells efficiently take in DNA in the form of aprecipitate with calcium phosphate. In electroporation, cells are placedin a solution containing DNA and subjected to a brief electrical pulsethat causes holes to open transiently in their membranes. DNA entersthrough the holes directly into the cytoplasm, bypassing the endocytoticvesicles through which they pass in the DEAE-dextran and calciumphosphate procedures (passage through these vesicles may sometimesdestroy or damage DNA). DNA can also be incorporated into artificiallipid vesicles, liposomes, which fuse with the cell membrane, deliveringtheir contents directly into the cytoplasm. In an even more directapproach, used primarily with plant cells and tissues, DNA is absorbedto the surface of tungsten microprojectiles and fired into cells with adevice resembling a shotgun.

Further methods for introducing nucleic acid molecules into cellsinvolve the use of viral vectors. Since viral growth depends on theability to get the viral genome into cells, viruses have devised cleverand efficient methods for doing it. One such virus widely used forprotein production is an insect virus, baculovirus. Baculovirusattracted the attention of researchers because during infection, itproduces one of its structural proteins (the coat protein) tospectacular levels. If a foreign gene were to be substituted for thisviral gene, it too ought to be produced at high level. Baculovirus, likevaccinia, is very large, and therefore foreign genes must be placed inthe viral genome by recombination. To express a foreign gene inbaculovirus, the gene of interest is cloned in place of the viral coatprotein gene in a plasmid carrying a small portion of the viral genome.The recombinant plasmid is cotransfected into insect cells withwild-type baculovirus DNA. At a low frequency, the plasmid and viralDNAs recombine through homologous sequences, resulting in the insertionof the foreign gene into the viral genome. Virus plaques develop, andthe plaques containing recombinant virus look different because theylack the coat protein. The plaques with recombinant virus are picked andexpanded. This virus stock is then used to infect a fresh culture ofinsect cells, resulting in high expression of the foreign protein. For areview of baculovirus vectors, see Miller [Bioessays 11:91-95 (1989)].Various viral vectors have also been used to transform mammalian cells,such as bacteriophage, vaccinia virus, adenovirus, and retrovirus.

As indicated, some of these methods of transforming a cell require theuse of an intermediate plasmid vector. U.S. Pat. No. 4,237,224 to Cohenand Boyer describes the production of expression systems in the form ofrecombinant plasmids using restriction enzyme cleavage and ligation withDNA ligase. These recombinant plasmids are then introduced by means oftransformation and replicated in unicellular cultures includingprocaryotic organisms and eucaryotic cells grown in tissue culture. TheDNA sequences are cloned into the plasmid vector using standard cloningprocedures known in the art, as described by Sambrook et al. [MolecularCloning: A Laboratory Manual, 2d Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)].

Host cells into which the nucleic acid encoding the triplet antigen hasbeen introduced can be used to produce (i.e. to functionally express)the triplet antigen.

The subject invention further provides a Treponema pallidum tripletantigen having an amino acid sequence selected from the group consistingof SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,and SEQ ID NO:12. Presently preferred embodiments of the triplet antigenare those represented by SEQ ID NO:9 and SEQ ID NO:12. These embodimentspresent the TP17 portion of the triplet before the TP15 portion of thetriplet, and higher antibody detection sensitivity is achieved withthese constructs. All constructs include a leader sequence (presentlypreferred is a ten amino acid leader sequence (tag 261), though othersuitable leaders could be substituted). All constructs further includehuman copper zinc superoxide dismutase (hSOD), a low immunogenicprotein, at the carboxy terminus. Eleven lysine residues in the hSODprovide sites for biotin attachment and HRP conjugation, and two cysteinresidues are mutated to serine (Cys 4 and Cys 112) to prevent interchainprotein polymerization [Hallewel et al., J Biol Chem 264:5260-5268(1989)]. This construct is thus optimized for in vitro diagnosis ofsyphilis infection. Other suitable low immunogenic proteins whichprovide similar sites for biotin attachment and HRP conjugation could besubstituted. In the presently preferred plasmid construction, thetriplet antigen is under the control of the T5 promoter.

While the specific details of an assay for detecting the presence ofTreponema pallidum antibodies in a sample are disclosed below, generallythe method comprises: contacting a sample with the Treponema pallidumtriplet antigen of the subject invention, wherein Treponema pallidumantibodies present in the sample bind to the Treponema pallidum tripletantigen forming an antibody/antigen complex; and detecting theantibody/antigen complex, thereby detecting the presence of theTreponema pallidum antibodies. For use in an assay format for thedetection of antibodies to Treponema pallidum, the antigen triplet maybe labeled with a detectable marker. Suitable markers include, forexample, enzymatic labels such as horseradish peroxidase or alkalinephosphatase, as well as fluorescent labels (such as fluorescein,rhodamine, and green fluorescent protein).

The assay format may also utilize biotin/avidin/streptavidin in theprovision of the triplet antigen bound to a solid phase. Suitable solidphases include, for example, any non-aqueous matrix to which the tripletantigen can be bound. Such solid phases are well known in theimmunoassay arts, and include, for example, polystyrene plates,polyacrylamides, glass, polysaccharides, polyvinyl alcohol andsilicones. Microslides, microwells, and microtips are all used as solidphases in immunoassays.

The assay format may involve direct detection of the antibody/antigencomplex (see FIG. 18), which can comprise: contacting theantibody/antigen complex with a second Treponema pallidum tripletantigen of the subject invention, wherein the second Treponema pallidumtriplet antigen is labeled with a detectable marker (HRP as shown). Thesecond Treponema pallidum triplet antigen binds to the antibody presentin the antibody/antigen complex forming an antigen/antibody/labeledantigen complex, which is then detected thereby detecting the presenceof Treponema pallidum antibodies in the sample.

The assay format may involve indirect detection of the antibody/antigencomplex (see FIG. 19), which can comprise: contacting theantibody/antigen complex with labeled anti-human antibody (monoclonalmouse anti-human antibody as shown). The labeled anti-human antibodybinds to the antibody present in the antibody/antigen complex forming anantigen/antibody/labeled anti-antibody complex, which is then detectedthereby detecting the presence of Treponema pallidum antibodies in thesample.

For all assays of the subject invention, the sample can be any suitablesample (for example, serum, plasma, and EDTA or heparin plasma) but ispreferably a serum sample.

The Treponema pallidum triplet antigen construct of the subjectinvention can thus be utilized as a component of a kit for detection ofTreponema pallidum antibodies. A kit is provided which comprises theTreponema pallidum triplet antigen construct, and additionally a secondTreponema pallidum triplet antigen construct labeled with a detectablemarker (an “enzyme conjugate” such as HRP-labeled Treponema pallidumtriplet antigen)(see above discussion of markers). The kit can alsocomprise suitable positive and/or negative controls, calibrationsamples, enzyme conjugates, substrate for enzyme conjugates (such as0-phenylenediamine), buffer solution, and washing solution.

The details of the construction of the triplet antigen and its use in anassay for detection of Treponema pallidum antibodies in a patient sampleare described below.

Synthetic Genes. T. pallidum outer membrane protein genes TP15, TP17,and TP47 were each synthesized based on amino acid sequence P16055(amino acid 19-141), P29722 (amino acid 23-156) and P29723 (amino acid21-434), respectively, published in the central database UniProt(http://www.uniprot.org). Human copper zinc superoxide dismutase (hSOD)gene was synthesized based on amino acid sequence P00441 (amino acid2-154), except two Cystein residues (amino acid 7 and 112) were mutatedto Serine to prevent polymerization. All four synthesized gene codonswere optimized for bacterial expression and each was inserted at theEcoR V site on host plasmid pUC57. The resulting plasmids, pUC57-15,pUC57-17, pUC57-45, and pUC57-hSOD, are shown in FIGS. 2-5,respectively.

The four synthetic genes do not bear stop codons and any internal Apa I,BamH I, Bgl II, EcoR I and Hind III sites that were used in subsequentsubcloning described below. Restriction enzyme sites, with or withouttag gene (261 sequence), were incorporated into the four syntheticgenes.

Expression Vector and TP Doublet Construction. Each of the TP genes frompUC57 was digested by Bgl II and Apa I (see FIG. 6 for resulting TPconstruct) and separately cloned into BamH I and Apa I sites of apreviously constructed expression vector, pB10G (see FIG. 8), whichcontained a T5 promoter, an ATG start site, and unique restriction sitesEcoR I, BamH I, Apa I and Hind III. This generated three expressionalvectors, pB10G-TP15, pB10G-TP17 and pB10G-TP47 (shown generically aspB10G-TP(1), pB10G-TP(2), and pB10G-TP(3) in FIGS. 9-11, respectively).Ligation of the compatible Bgl II ends of the gene fragments and theBamH I ends on the plasmids eliminated the Bgl II/BamH I restrictionenzyme sites in all three constructs. The TP doublet was created bysubcloning. To construct a TP doublet subclone (see FIG. 13), a DNA geneinsert was produced by PCR reaction using the second antigen expressionvector as template and a pair of forward and reverse primers. Theforward primer covered the EcoR I site, located in the T5 promoterregion, upstream of the TP gene. The reverse primer matched the end ofthe TP gene and converted the Apa I site to a Bgl II site. This PCRproduct was then digested with EcoR I/Bgl II (see FIG. 12 for resultingTP-1 construct) and cloned into another antigen expression vector whichwas cut open with EcoR I and BamH I. Likewise, ligation of thecompatible Bgl II on the insert and the BamH I on the host eliminatedboth restriction enzyme sites.

TP Triplet Construction. To produce six final triplet fusion genes, fourTP doublet vectors, pB10G-TP17-TP47, pB10G-TP15-TP47, pB10G-TP47-TP15and pB10G-TP17-TP15, were used as the host and the third TP gene waseither added at the 5′end of the TP doublet or added at the 3′end of theTP doublet. The TP(1-2) double vector is shown generically in FIG. 13and the TP(3) construct for insertion into the doublet vector is shownin FIG. 14. To add the insert at the 3′ end, a DNA gene insert wasproduced by PCR reaction using the third antigen expression vector astemplate and a pair of forward and reverse primers. The forward primermatched the 5′ end of TP gene and contained a Apa I site. The reverseprimer matched the 3′ end of TP gene and contained a Hind III site. ThisPCR product was then digested with Apa I/Hind III and cloned into thedoublet expression vector which was cut open with the appropriaterestriction enzymes. Triplets pB10G-TP15-TP17-TP47, pB10G-TP17-TP15-TP47and pB10G-TP47-TP17-TP15 were made by adding the third TP gene at thedoublet 5′end, while triplets pB10G-TP15-TP47-TP17, pB10G-TP47-TP15-TP17and pB10G-TP17-TP47-TP15 were made by adding the third TP gene at thedoublet 3′ end. PCR primers used in creating the doublets and tripletsare listed in Table 4, PCR Primers, Group A. This triplet construct isshown generically in FIG. 15.

TP Triplet with SOD fusion Construction. The TP triplet with aC-terminal SOD fusion tag (shown generically in FIG. 17) was createdthrough “two-round” PCR and cloning. “Two-round PCR” was performed tolink the C-terminal TP3 with SOD (see FIG. 7 for SOD construct) and tointroduce restriction enzyme sites for cloning. The first round of PCRwas composed of two separate PCR reactions, with the reverse primer(Table 4, PCR Primers, Group B) in one PCR reaction complementing theforward primer (Table 4, PCR Primers, Group C) in the other PCRreaction. In the second round of PCR, the two products from the firstround PCR were combined and amplified with a third set of nested primers(Table 4, PCR Primers, Group D). The forward primer matched the TP3sequence and contained a unique restriction enzyme (RE) site. Thereverse primer matched the 3′end SOD. Two stop codons and a Hind IIIsite created a new DNA insert (see FIG. 16). To fuse the SOD gene to theexisting T. pallidum triplet, the new PCR product was digested anddirectly cloned into a pB10G-TP triplet plasmid digested at the designedunique restriction site and the Hind III site to yieldpB10G-TP(1-2-3)-SOD (see FIG. 17). As used herein, TP1, TP2, and TP3represent the three T. pallidum antigens, TP15, TP17, and TP47, in anyorder. Thus, TP1 could be TP15, TP2 could be TP17, and TP3 could beTP47. Other combinations include: TP1 could be TP15, TP2 could be TP47,and TP3 could be TP17; TP1 could be TP17, TP2 could be TP15, and TP3could be TP47; TP1 could be TP17, TP2 could be TP47, and TP3 could beTP15; TP1 could be TP47, TP2 could be TP15, and TP3 could be TP17; andTP1 could be TP47, TP2 could be TP17, and TP3 could be TP15.

All PCR amplifications were performed with Taq polymerase using astandard sequence of 35 PCR cycles: 95° C. (15 sec), 55° C. (20 sec),72° C. (30 sec). Nucleotide sequences of the PCR primers are listed inTable 4. Usage of each primer in creating particular triplets isindicated. All PCR products were purified following a Qiagen PCR kitprotocol. All restriction enzymes were purchased from New EnglandBiolabs. Plasmids were prepared using Qiagen DNA Miniprep kits. All sixtriplet coding regions were DNA sequenced (SEQ ID NOs:1-6) and aminoacid sequences translated (SEQ ID NOs:7-12).

The assay of the subject invention provides for the measurement ofantibodies to three T. pallidum antigens, TP15, TP17 and TP47. The assayis performed on antigen precoated microtiter plates. Samples are addedto the microtiter plate wells and incubated. T. pallidum IgG/IgMspecific antibodies, if present, will bind to and become immobilized bythe antigen pre-coated on the wells. The bound antibodies were detectedeither in a direct conjugated antigen sandwich format (see FIG. 18), orin an indirect format detected by conjugated anti-human IgG and IgM (seeFIG. 19).

More particularly, recombinant TP triplets were coated passively on anELISA high-binding plate well surface as capture antigen. The plate wasthen blocked with 1% BSA/PBS to cover all unbound well surfaces.Syphilis infected patient's serum or plasma was added in wells andincubated for a first incubation period, enabling T. pallidum antibody(IgG, IgM, and IgA) in the sample to react with the precoated tripletantigens. Unbound materials were washed away after the first incubation.For the direct assay, HRP conjugated recombinant TP triplet was thedetector and was added into the wells and incubated for a secondincubation period. After the second incubation, unbound tripletconjugates were washed away. The formed antigen—human T. pallidumantibody (IgG/IgM)—antigen complex was measured by adding peroxidasesubstrate solution, then the reaction was stopped after 30 minutes andoptical density was recorded for analysis. For the indirect assay, anHRP conjugated mouse monoclonal anti-human IgG and HRP conjugated mousemonoclonal anti-human IgM mixture was the detector and was added intothe wells and incubated for the second incubation period. After thesecond incubation, unbound conjugates were washed away. The formedanti-human IgG/IgM —human T. pallidum antibody (IgG/IgM)—antigen complexwas measured by adding peroxidase substrate solution, then the reactionwas stopped after 30 minutes and optical density was recorded foranalysis.

The engineered recombinant T. pallidum triplet has a 10 amino acidleader sequence (tag 261) at the N-terminus and two to four amino acidlinkers between each TP antigen. The tag 261 sequence was derived fromhuman placenta growth factor (P1GF). The human copper zinc superoxidedismutase (hSOD) is incorporated at the C-terminus of the T. pallidumantigen triplet to form a fusion protein.

hSOD has been used previously in various recombinant antigen fusions indiagnostic assays for infectious pathogens such as HCV, HIV etc. hSOD isa small size, low immunogenic human endogenous protein, which has 153amino acids with a molecular weight of about 16 kD. The 11 lysineresidues in hSOD provide extra conjugation site for biotinylation andHRP conjugation.

Example I ELISA Assay Reagents, Format, and Protocol

Assay Reagents:

-   -   96 well microtiter high-binding plate (Costar)    -   Coating buffer (10 mM phosphate, 2 mM EDTA, pH 7.0), blocking        buffer (1% BSA in PBS, pH 7.0), washing solution (PBS with 0.05%        twenn-20), sample buffer (Blocker Casein in PBS with 0.05%        tween-20, Pierce)    -   ELISA specimen and conjugate diluent: Blocker Casein in PBS from        Pierce. Tween 20 was added to a final of 0.05% before use. In        the direct assay, hSOD lysate was added together with HRP        conjugated TP triplet.    -   Purified recombinant TP triplet fusions, their sequences were        validated by DNA sequencing (see SEQ ID NOs:1-6). Proteins were        expressed in prokaryotic E. coli cell. Protein purity was        validated to be greater than 87% by SDS PAGE.    -   HRP (horseradish peroxidase) conjugated recombinant TP triplet.    -   150 ng/mL, Phosphate, pH=7.2, Molarity=50 mM HRP conjugate        reagent buffer contains: H2O, K2HPO4 (anhydrous), KH2PO4        (anhydrous), NaCl, BSA liquid, KFeCN, ANS, Tween-20, Anti-foam        204, ProClin 950    -   HRP (horseradish peroxidase) conjugated mouse monoclonal        anti-261 tag    -   HRP (horseradish peroxidase) conjugated mouse monoclonal        anti-human IgG    -   HRP (horseradish peroxidase) conjugated mouse monoclonal        anti-human IgM    -   HRP (horseradish peroxidase) substrate tablet        (O-Phenylenediamine-2HCl) and solution and stop solution are        components from Ortho-Clinical Diagnostics general ELISA        products.    -   Specimens: Syphilis Positive plasma, Syphilis Negative plasma,        SeraCare Syphilis mixed titer panel (PS C202) and Zeptometrix        Syphilis mixed titer panel (K-ZMC002)

The ELISA Assay Format is shown in FIGS. 18 and 19, both a direct assayformat and an indirect assay format.

ELISA Assay Protocol: Plate coating: 1) add 100 uL/well coating solutioncontaining 2 ug/mL of TP triplet fusion at 25° C. for 18 hrs. 2) Wellswere washed once with washing buffer and 290 uL/well blocking bufferwere added for 1 hr/25° C. blocking. 3) After blocking buffer aspirated,plates were dried greater than 4 hrs in a low humidity incubator. 4)Plate was pouched in an air-proof sealed bag until use.

Direct Assay Protocol: Assay: 1) Add 50 uL Casein (PBS) specimen diluentand 50 uL specimen (or control) to each well. Plate was incubated for 30min at 37° C. with shaking. 2) After 6 times wash with washing solution;add 100 uL HRP conjugated TP triplet fusion diluted in Casein (PBS) toeach well. Plate was incubated for 30 min at 37° C. with shaking. 3)After 6 times wash, add 100 uL OPD substrate and incubate in dark for 30min at 25° C. 4) Add 25 uL stop solution and read optical density (OD)at 492 nm.

Indirect Assay Protocol: Assay: 1) Add 90 uL Casein (PBS) specimendiluent and 10 uL specimen (or control) to each well. Plate wasincubated for 15 min at 37° C. with shaking. 2) After 6 times wash withwashing solution; add 100 uL HRP conjugate mixture containing HRP-mousemonoclonal anti-human IgG and HRP-mouse monoclonal anti-human IgMdiluted in casein (PBS) to each well. Plate was incubated for 15 min at37° C. with shaking. 3) After 6 times wash, add 100 uL OPD substrate andincubate in dark for 30 min at 25° C. 4) Add 25 uL stop solution andread optical density (OD) at 492 nm.

Example II Evaluation of Triplet Constructs

ELISA Reaction: (1) Wells were coated with a serial dilution of six TPtriplets and post-coated with 1% BSA in PBS. (2) Add 100 ul HRPconjugated mouse monoclonal anti-261 tag diluted in Casein (PBS) toantigen precoated wells, and incubate at 37° C. for 15 minutes withshaking. (2) Wash 6 times, add 100 uL OPD substrate solution, andincubate at RT for 15 min in dark. (4) Add 25 uL 4N sulfuric acid stopsolution and read at 490 nm.

Results shown in Table 1 were ODs. Proposed TP Triplet coatingconcentrations were derived from calculation to calibrate antigenquantity immobilized on the plate and used in plate coating in theantibody assay evaluation.

ELISA Reaction: (1) Wells were coated with six TP triplets at aconcentration defined in Table-1, and post-coated with 1% BSA in PBS.(2) Add 50 ul Casein (PBS) and 50 ul panel specimens to antigenprecoated wells, and incubate at 37° C. for 15 minutes with shaking. (3)Wash 6 times, add 100 ul HRP conjugated TP triplet antigens, andincubate at 37° C. for 15 minutes with shaking. HRP conjugated antigenis the antigen coated on the plates. (4) Wash 6 times, add 100 uL OPDsubstrate solution, and incubate at RT for 15 min in dark. (4) Add 25 uL4N sulfuric acid stop solution and read at 490 nm.

Results shown in Table 2 were S/C values. S is OD signal, C is cut-off,equals 5 times of an average OD given by three negative controls.

ELISA Reaction: (1) Add 90 ul Casein and 10 ul panel sample (2 foldserial diluted in normal human plasma) to antigen precoated wells, andincubate at 37° C. for 15 minutes with shaking. (2) Wash 6 times, add100 ul conjugate mixture containing HRP mouse monoclonal anti-human IgGand monoclonal anti-human IgM, and incubate at 37° C. for 15 minuteswith shaking. (3) Wash 6 times, add 100 uL OPD substrate solution, andincubate at RT for 15 minutes in dark. (4) Add 25 uL 4N sulfuric acidstop solution and read at 490 nm.

Results shown in Table 3 were final dilution of ZeptoMetrix panelspecimen with normal human plasma (1:X, X=), at which dilution thespecimens were determined to be positive (signal over cut-off >1). Thecut-off is 5 times of an average OD given by three negative controls.

Example III Details of VITROS® Syphilis TPA Test

The principles of the VITROS® Syphilis TPA test using the TP15-TP17-TP47triplet construct are as described above and as shown in FIG. 1. A kitis provided which includes a reagent pack and a calibrator. The reagentpack contains: 100 coated wells (streptavidin, bacterial; binds ≥2 ngbiotin/well); 13.1 mL biotinylated antigen reagent (biotin-recombinantTP antigens 0.15 ug/mL) in buffer with bovine gamma globulin, bovineserum albumin, and antimicrobial agent; and 20.4 mL conjugate reagent(HRP-recombinant TP antigens, 0.15 ug/mL) in buffer with bovine serumalbumin and antimicrobial agent). The calibrator contains VITROS®Syphilis TPA Calibrator (human syphilis IgG positive plasma, 2.2 mL)with antimicrobial agent. The test uses 25 uL of calibrator for eachdetermination.

Suitable specimens for use with the test are serum, heparin plasma, EDTAplasma, and citrate plasma. The test uses 25 uL of sample (specimen) foreach determination.

The test also uses signal reagent (such as VITROS® ImmunodiagnosticProducts Signal Reagent), wash reagent (such as VITROS® ImmunodiagnosticProducts Universal Wash Reagent), and quality control materials (such asVITROS® Immunodiagnostic Products Syphilis TPA Controls).

The test uses a 16-minute first incubation period, and an 8-minutesecond incubation period, with a time for first result of 34 minutes.The test is performed at 37° C.

Results are automatically calculated by the VITROS® Immunodiagnostic andVITROS® Integrated Systems, and represent “signal for testsample”/“signal at cutoff (cutoff value)”. Samples with results of <0.80will be flagged as “negative”, samples with results ≥0.80 and <1.20 willbe flagged as “borderline”, and samples with results ≥1.20 will beflagged as “reactive”. Negative indicates no active or previousinfection with Treponema pallidum; borderline indicates the test isunable to determine if Treponema pallidum infection has occurred, andthe sample should be re-tested; and reactive indicates active orprevious infection with Treponema pallidum.

Example IV Performance Characteristics of the VITROS® Syphilis TPA Test

Referring to Table 5, initial sensitivity and specificity was assessedon a population of 4290 samples using the VITROS® Syphilis TPA test anda commercially available immunoassay (“IA 1”) for antibodies toTreponema pallidum. An initial analysis in the VITROS® Syphilis TPA testgave an initial specificity, including borderline samples (4015/4016) of99.98% (exact 95% Cl 99.9-100.0%). Initial sensitivity, includingborderline samples (266/274) was 97.08% (exact 95% Cl 94.3-98.7%). One(0.025%) sample was borderline in the VITROS® Syphilis TPA test. Thecommercially available test did not have a borderline region.

Referring to Table 6, relative specificity and sensitivity afterresolution of uninterpretable samples was assessed. This includedsamples where there was a difference in classification from thecommercial test (reactive/negative)(defined as “discordant”). Samplesthat resulted in discordant or borderline results (either in the VITROS®or IA 1 test) were further tested to determine relative sensitivity andspecificity. A total of 9 discordant and borderline samples were furthertested by first repeating the VITROS® Syphilis TPA test in duplicate. Atotal of 9 discordant and borderline samples remained discordant with IA1 after repeat testing in the VITROS® Syphilis TPA test. The 9 sampleswere also tested in up to 4 additional commercially available assays forantibodies to Treponema pallidum. The median VITROS® Syphilis TPA resultwas then compared to the consensus classification of the other 4commercially available tests. Using this algorithm, 8 samples wereresolved as syphilis antibody negative and one sample remainedborderline in the VITROS® Syphilis TPA test. After resolution ofdiscordant results, the relative specificity of the VITROS® Syphilis TPAtest to the IA 1 test was calculated (4023/4024) as 99.98% (exact Cl99.9-100.0%) and relative sensitivity (266/266) as 100% (exact Cl98.6-100.0%).

Referring to Table 7, 149 samples containing potentially cross-reactingsub-groups were tested in the VITROS® Syphilis TPA test and in acommercially available test (EIA 1). The sub-groups included: HAV IgGand IgM, HBV IgG and IgM, HCV IgG and IgM, EBV IgG and IgM, anti-HSV IgGand IgM, anti-HIV 1/2 IgG and IgM, CMV IgG and IgM, Rubella IgG and IgM,ANA/SLE, Borrelia burgdorferi infection (European and US strain),Toxoplasma gondii infections IgG and IgM, heterophilic antibodies/HAMAand Rheumatoid factor. The specificity (137/137) was 100.0% (95% Cl97.3-100.0%) and sensitivity (12/12) was 100.0% (95% Cl 73.5%-100.0%).No discordant samples were observed and all results were in line withthe expected clinical performance in the commercially available test.Thus, none of the samples was found to cross react with the VITROS®Syphilis TPA assay to cause any mis-classification of results.

Precision on the VITROS® ECi/ECiQ Immunodiagnostic System was evaluated.Two replicates each of 4 patient sample pools and 4 control samples weretested on 2 separate occasions per day on at least 20 different days.The experiment was performed using 2 reagent lots on two differentsystems. Precision on the VITROS® 3600 Immunodiagnostic System and theVITROS® 5600 Integrated System was also evaluated. Two replicates eachof 4 patient sample pools and 4 control samples were tested on 2separate occasions per day on at least 20 different days. The experimentwas performed using 1 reagent lot on each system. Results showedprecision for samples at the cut off up to strong positives averaged1.6% (range 0.9-3.2%) within run, 4.8% (range 2.7-9.0%) withincalibration, and 4.6% (range 2.1-9.0%) within lab. The VITROS® SyphilisTPA test thus gives excellent precision across the borderline andreactive ranges, on all VITROS® systems.

Referring to Table 8, the VITROS® Syphilis TPA test was evaluated forinterference. Of the compounds tested, none was found to interferencewith the clinical interpretation of the test at the concentrationindicated.

The VITROS® Syphilis TPA test was also evaluated with two sets ofproficiency samples from CAP and NEQAS and two commercially availableperformance panels (Zeptometrix and BBI-Seracare). 100% agreement wasobtained with these proficiency samples and performance panels.

Samples types were also evaluation on the VITROS® Syphilis TPA test.Five normal donor samples were collected as serum (SST, clot activatorand on the clot glass tubes), as heparin plasma (lithium and sodium),EDTA plasma and citrate plasma. From five other donors, 50 mL of wholeblood was collected. This was spiked with a syphilis reactive plasma anddispensed into the same type of tubes as mentioned above to mimicsyphilis reactive donors. Bias between sample type was assessed. Nolarge differences were observed between sample types compared to serum.For citrate samples, the recovery compared to serum was lower due to thedilutional effect of the citrate. A stability study using these samplesdemonstrated that samples can be stored for 7 days at 2-8° C. and 4weeks at −20° C. without significant loss of dose results or change inclinical classification.

Taking all performance characteristics into account, the VITROS®Syphilis TPA test combines good analytical and clinical performance withthe operational simplicity of a rapid automated continuous random accessimmunoassay.

While particular embodiments of the invention have been shown, it willbe understood, of course, that the invention is not limited thereto,since modifications may be made by those skilled in the art,particularly in light of the foregoing teachings. Reasonable variationand modification are possible within the scope of the foregoingdisclosure of the invention without departing from the spirit of theinvention.

TABLE 1 Calibration Coated Six TP Triplet Antigens on Plate Proposed TPPlate coated TP OD signal probed by HRP anti-261 (2 ng/mL) Tripletcoating Triplet [ug/ml] 4.00 2.00 1.00 0.50 0.25 0.13 [ug/mL]TP15-17-47-SOD over 3.006 2.372 1.490 0.914 0.613 1.00 TP15-47-17-SODover 2.916 2.106 1.356 0.843 0.566 1.20 TP17-15-47-SOD over 3.144 3.1932.508 1.494 0.891 0.50 TP17-47-15-SOD over 1.454 0.914 0.580 0.346 0.2632.80 TP47-15-17-SOD over 3.310 2.481 1.950 1.209 0.713 0.65TP47-17-15-SOD over 3.523 3.626 3.489 2.434 1.426 0.25

TABLE 2 Evaluation of Syphilis Reactive Specimens by Direct ELISA Plate:TP Triplet Coated SeraCare Syphilis mixed titer panel (PS C202)-(S/CValues) ID [ug/ml] #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 TP15-17-47-SOD 1.003.0 5.4 1.1 0.0 2.6 2.6 8.0 1.3 7.7 4.0 TP15-47-17-SOD 1.20 4.5 7.2 2.70.0 1.8 4.5 14 4.0 7.8 6.1 TP17-15-47-SOD 0.50 4.9 10 3.4 0.0 3.0 4.9 156.7 15 6.7 TP17-47-15-SOD 2.80 3.1 8.8 3.0 0.0 2.6 4.9 4.7 5.6 15 6.7TP47-15-17-SOD 0.65 4.4 9.7 1.9 0.0 3.1 4.9 17 3.4 15 5.4 TP47-17-15-SOD0.25 4.9 10 3.3 0.0 4.2 4.9 15 6.0 15 6.7 Plate: TP Triplet Coated ID[ug/ml] #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 TP15-17-47-SOD 1.00 2.13.3 3.2 2.9 5.3 0.0 3.1 2.6 2.8 3.9 TP15-47-17-SOD 1.20 3.0 6.1 6.9 8.16.9 0.0 3.2 7.0 4.0 2.8 TP17-15-47-SOD 0.50 6.3 6.7 11 7.5 10 0.0 4.6 164.5 10 TP17-47-15-SOD 2.80 5.6 4.0 14 6.2 10 0.0 3.6 10 4.2 5.3TP47-15-17-SOD 0.65 4.9 6.1 11 6.6 10 0.0 2.5 5.0 5.8 5.6 TP47-17-15-SOD0.25 7.0 6.7 11 9.7 13 0.0 3.5 12 3.9 9.0

TABLE 3 Evaluation of Plate Coating Antigen Sequence Combination Plate:TP Triplet Coated ZeptoMetrix Mixed Titer Syphilis Panel (K-ZMC002) ID[ug/ml] #1 #2 #3 #4 #5 #6 #7 #8 TP15-17-47-SOD 1.00 2 2 2 4 8 8 8 2TP15-47-17-SOD 1.20 4 2 2 8 4 16 8 2 TP17-15-47-SOD 0.50 16 4 2 256 3264 256 8 TP17-47-15-SOD 2.80 8 2 2 32 4 8 64 2 TP47-15-17-SOD 0.65 16 24 64 32 16 256 8 TP47-17-15-SOD 0.25 32 4 4 256 16 64 256 8 Plate: TPTriplet Coated ID [ug/ml] #9 #10 #11 #12 #13 #14 #15 TP15-17-47-SOD 1.002 32 2 2 4 256 8 TP15-47-17-SOD 1.20 2 16 2 2 4 32 8 TP17-15-47-SOD 0.5032 256 8 64 32 256 32 TP17-47-15-SOD 2.80 2 256 2 8 8 256 16TP47-15-17-SOD 0.65 16 256 4 16 16 256 32 TP47-17-15-SOD 0.25 16 256 832 16 256 64

TABLE 4 PCR Primers Primers used in creating triplet of 15- 17- 15- 47-17- 47- Primer 17- 15- 47- 15- 47- 17- Group Name Direction 47 47 17 1715 15 Comments A F4-ER Forward yes yes yes yes yes yes EcoR I, vectorsequence Apa-TP15 Forward yes Apa -(TP15)... Apa-TP17 Forward yes yesApa -(TP17)... TP15-BG Reverse yes ...TP15-gly-pro- Bgl II TP15-BG2Reverse yes yes ...TP15-gly-Bgl II TP17-BG Reverse yes yes yes yes...TP17-gly-pro- Bgl II TP47-BG2 Reverse yes yes ...TP47-gly-pro- Bgl IIB F-15 Forward yes yes 50 bp before SacI in TP15 R-15S Reverse yes yesTP15(3′)-SOD(5′) T17-BH Forward yes yes 95 bp before Eag I in TP17 R-17SReverse yes yes TP17(3′)-SOD(5′) F-47b Forward yes yes 50 bp before Xmain TP47 R-47S Reverse yes yes TP47(3′)-SOD(5′) C F-15S Forward yes yesTP15(3′)-SOD(5′) F-17S Forward yes yes TP17(3′)-SOD(5′) F-47S Forwardyes yes TP47(3′)-SOD(5′) RS Reverse yes yes yes yes yes yesdownstream of SOD gene D T15-Sac Forward yes yes (TP15)...SacI... TP17-Forward yes yes (TP17)...EagI... EAG T47-XMA Forward yes yes(TP47)...XmaI... RS-H3 Reverse yes yes yes yes yes yes SOD(3′)-StopStop-Hind III Group Name Primer Sequence (5′-3′) A F4-ERSEQ ID NO: 13: cacaGAATTCATTAAAGAGGAGAAATTAAC Apa-TP15SEQ ID NO: 14: tgtctGGGCCCAGCTTTTCTAGTATTCCGA Apa-TP17SEQ ID NO: 15: tgtctGGGCCCGTGAGCTGCACCACGGT TP15-BGSEQ ID NO: 16: agctggAGATCTCGGGCCGCGAGAGATAATGGCTTCTT TP15-BG2SEQ ID NO: 17: gctggAGATCTACCGCGAGAGATAATGGCTTCTT TP17-BGSEQ ID NO: 18: agctggAGATCTCGGGCCTTTCTTGGTTTTCTTCAGAACGTA TP47-BG2SEQ ID NO: 19: agctggAGATCTTGGACCCTGCGCCACCACTTTCGCG B F-15SEQ ID NO: 20: CGCGACCGTGAGCTCTCAGAGTTTT R-15SSEQ ID NO: 21: CAGCACGCTGACGGCTTTGGTCGCgagGCGAGAGATAATGGCTTCTTTTTCGCCT17-BH SEQ ID NO: 22: GTGAGCTGCACCACGGT R-17SSEQ ID NO: 23: CAGCACGCTGACGGCTTTGGTCGCgagTTTCTTGGTTTTCTTCAGAACGTAAAF-47b SEQ ID NO: 24: GGTTAGCGATCAGGCCGT R-475SEQ ID NO: 25: CAGCACGCTGACGGCTTTGGTCGCgagCTGCGCCACCACTTTCGCGCGC C F-15SSEQ ID NO: 26: GGCGAAAAAGAAGCCATTATCTCTCGCctcGCGACCAAAGCCGTCAGCGTGCTGF-17SSEQ ID NO: 27: TTTACGTTCTGAAGAAAACCAAGAAActcGCGACCAAAGCCGTCAGCGTGCTGF-475 SEQ ID NO: 28: GCGCGCGAAAGTGGTGGCGCAGctcGCGACCAAAGCCGTCAGCGTGCTGRS SEQ ID NO: 29: TGCAGTCGACGGGCCCGGGAT D T15-SacSEQ ID NO: 30: CGCGACCGTGAGCTCTCAGAGTTTT TP17-EAGSEQ ID NO: 31: CCCTGCCGGCCGCAGATTGT T47-XMASEQ ID NO: 32: GGATTTCACCCCGGGTACCGAATATA RS-H3SEQ ID NO: 33: agccAAGCTTcattaCTGGGCGATACCAATAACGCCA

TABLE 5 VITROS Syphilis TPA Test Reactive Borderline Negative Total IA 1Reactive 266 0 8 274 Negative 0 1 4015 4016 Totals 266 1 4023 4290

TABLE 6 VITROS Syphilis TPA Test Reactive Borderline Negative Total IA 1Reactive 266 0 0 266 Negative 0 1 4023 4024 Totals 266 1 4023 4290

TABLE 7 VITROS Syphilis TPA Test Reactive Borderline Negative Total EIA1 Reactive 12 0 0 12 Negative 0 0 137 137 Totals 12 0 137 149

TABLE 8 Compound Concentration Azide (sodium) 20 mg/dL  3.06 mmol/LBilirubin 20 mg/dL 0.342 mmol/L Biotin 1000 ng/dL  40.8 nmol/L BSA (HighProtein) 5 g/dL (total ~12 g/dL) N/A Cholesterol 250 mg/dL N/AHemoglobin 500 mg/dL 0.155 mmol/L (hemolysate) Intralipid 850 mg/dL N/ATriolein 3000 mg/dL 33.96 mmol/L

1.-27. (canceled)
 28. A Treponema pallidum triplet antigen comprising anamino acid sequence selected from the group consisting of SEQ ID NO:7,SEQ ID NO:8, and SEQ ID NO:10.
 29. The Treponema pallidum tripletantigen of claim 28 comprising an amino acid sequence as shown in SEQ IDNO:7.
 30. The Treponema pallidum triplet antigen of claim 28 comprisingan amino acid sequence as shown in SEQ ID NO:8.
 31. (canceled)
 32. TheTreponema pallidum triplet antigen of claim 28 comprising an amino acidsequence as shown in SEQ ID NO:10.
 33. (canceled)
 34. (canceled)
 35. TheTreponema pallidum triplet antigen of claim 28 labeled with a detectablemarker.
 36. The Treponema pallidum triplet antigen of claim 35 whereinthe detectable marker is horseradish peroxidase.
 37. A method ofdetecting the presence of Treponema pallidum antibodies in a sample, themethod comprising: contacting the sample with a first antigen which isimmobilized on a solid support and which comprises the Treponemapallidum triplet antigen of claim 29, wherein Treponema pallidumantibodies present in the sample bind to the Treponema pallidum tripletantigen forming an antibody/antigen complex; and detecting theantibody/antigen complex, thereby detecting the presence of Treponemapallidum antibodies, wherein the antibody/antigen complex is detected by(i) contacting the antibody/antigen complex with a Treponema pallidumtriplet antigen which comprises SEQ ID NO: 7, is labeled with adetectable marker, and binds to the antibody present in theantibody/antigen complex forming an antigen/antibody/labeled antigencomplex, and detecting the antigen/antibody/labeled antigen complex; or(ii) contacting the antibody/antigen complex with a labeled anti-humanantibody, wherein the labeled anti-human antibody binds to the antibodypresent in the antibody/antigen complex forming anantigen/antibody/labeled anti-antibody complex; and detecting theantigen/antibody/labeled anti-antibody complex. 38.-41. (canceled)
 42. Akit for detection of Treponema pallidum antibodies, the kit comprising:the Treponema pallidum triplet antigen of claim 28; and a secondTreponema pallidum triplet antigen of claim 28, the second Treponemapallidum triplet antigen labeled with a detectable marker.
 43. The kitof claim 42 further comprising positive or negative controls.
 44. Thekit of claim 42 further comprising calibration samples.
 45. The kit ofclaim 42 further comprising one or more of enzyme conjugate, substratefor enzyme conjugate, buffer solution, and washing solution.
 46. Amethod of detecting the presence of Treponema pallidum antibodies in asample, the method comprising: contacting the sample with a firstantigen which is immobilized on a solid support and which comprises theTreponema pallidum triplet antigen of claim 30, wherein Treponemapallidum antibodies present in the sample bind to the Treponema pallidumtriplet antigen forming an antibody/antigen complex; and detecting theantibody/antigen complex, thereby detecting the presence of Treponemapallidum antibodies, wherein the antibody/antigen complex is detected by(i) contacting the antibody/antigen complex with a Treponema pallidumtriplet antigen which comprises SEQ ID NO: 8, is labeled with adetectable marker, and binds to the antibody present in theantibody/antigen complex forming an antigen/antibody/labeled antigencomplex, and detecting the antigen/antibody/labeled antigen complex; or(ii) contacting the antibody/antigen complex with a labeled anti-humanantibody, wherein the labeled anti-human antibody binds to the antibodypresent in the antibody/antigen complex forming anantigen/antibody/labeled anti-antibody complex; and detecting theantigen/antibody/labeled anti-antibody complex.
 47. A method ofdetecting the presence of Treponema pallidum antibodies in a sample, themethod comprising: contacting the sample with a first antigen which isimmobilized on a solid support and which comprises the Treponemapallidum triplet antigen of claim 32, wherein Treponema pallidumantibodies present in the sample bind to the Treponema pallidum tripletantigen forming an antibody/antigen complex; and detecting theantibody/antigen complex, thereby detecting the presence of Treponemapallidum antibodies, wherein the antibody/antigen complex is detected by(i) contacting the antibody/antigen complex with a Treponema pallidumtriplet antigen which comprises SEQ ID NO: 10, is labeled with adetectable marker, and binds to the antibody present in theantibody/antigen complex forming an antigen/antibody/labeled antigencomplex, and detecting the antigen/antibody/labeled antigen complex; or(ii) contacting the antibody/antigen complex with a labeled anti-humanantibody, wherein the labeled anti-human antibody binds to the antibodypresent in the antibody/antigen complex forming anantigen/antibody/labeled anti-antibody complex; and detecting theantigen/antibody/labeled anti-antibody complex.